Ligand specific to b2-glycoprotein I and use thereof

ABSTRACT

The cholesterol derivative, represented by the following formula (1):  
                 
 
     wherein R represents a C3-C23 saturated or unsaturated aliphatic hydrocarbon residue having an optional substituent, and, to the cholesterol backbone, —OH, —CHO, —COOH, —OOH, or an epoxy group may be added, specifically binds to β 2 -glycoprotein. By use of the derivative, β 2 -glycoprotein or a similar substance can be assayed in a very practical manner. Through the assay, a disease can be detected in a very practical manner.

TECHNICAL FIELD

[0001] The present invention relates to a ligand specific toβ₂-glycoprotein I (β₂-GPI) and derivatives of the ligand; to an assaymethod for β₂-GPI making use of any of the ligand and derivatives; to anassay method for an antibody recognizing a ligand-β₂-GPI complex; and toa method for detecting a disease.

BACKGROUND ART

[0002] β₂-GPI is a major antigen which is present in patients withantiphospholipid syndrome (APS) and is recognized by “anantiphospholipid antibodies.” β₂-GPI is known to specifically bind tooxidized low-density lipoprotein (oxidized LDL; oxLDL), but not tonon-oxidized (native) low-density lipoprotein (LDL). PCT InternationalPatent Publication (pamphlet) WO 95/9363 discloses an oxLDL assay methodbased on a specific binding property with respect to β₂-GPI, a kit fordiagnosing an arteriosclerotic disease employing the assay method, etc.

[0003] However, the portion of the oxLDL structure which β₂-GPIrecognizes for binding has not been identified.

[0004] Therefore, identification of the portion of the oxLDL structureto which β₂-GPI specifically binds would realize an assay of β₂-GPI or asimilar assay by use of an easier and simpler system employing asubstance including the portion. In addition, handling and storage ofthe reagents used in the assay would be further facilitated, leading toprovision of constant-quality assay reagents, assay kits, etc. at lowcost.

[0005] In view of the foregoing, the present inventors have carried outextensive studies in order to provide a substance having a structurewhich specifically binds to β₂-GPI; a very practical assay method forβ₂-GPI or the like making use of the substance; and a very practicalmethod for detecting a disease on the basis of the assay method. Theinventors have isolated a substance having a structure whichspecifically binds to β₂-GPI, and based on the isolated substance, haveestablished an assay method for β₂-GPI or the like as well as a methodfor detecting a disease employing the assay method. The presentinvention has been accomplished on the basis of these findings.

DISCLOSURE OF THE INVENTION

[0006] Accordingly, the present invention is directed to a cholesterolderivative represented by the following formula (1) (hereinafterreferred to as the derivative of the present invention):

[0007] wherein R represents a C3-C23 saturated or unsaturated aliphatichydrocarbon residue having an optional substituent, and, to thecholesterol backbone, —OH, —CHO, —COOH, —OOH, or an epoxy group may beadded.

[0008] The aforementioned R preferably has one or more substituentsselected from the group consisting of —COOH, —OH, —CHO, an oxo group,and an epoxy group.

[0009] R is preferably a C3-C23 saturated or unsaturated fatty acidresidue which may have one or more oxo groups. More preferably, R is agroup represented by HOOC—R′— (wherein R′ represents a C2-C22 saturatedor unsaturated aliphatic hydrocarbon residue which may have one or moreoxo groups).

[0010] R′ is preferably a linear-chain aliphatic hydrocarbon residuehaving one oxo group.

[0011] In relation to specific examples of the derivative of the presentinvention, the invention is also directed to cholesterol derivativesrepresented by the following formulas (2) to (7).

[0012] The present invention is also directed to a solid phase on whichthe derivative of the present invention has been immobilized(hereinafter referred to as the “solid phase of the present invention”).

[0013] The present invention is also directed to an assay method forβ₂-GPI, characterized in that the method includes at least the followingsteps (hereinafter referred to as “assay method 1 of the presentinvention”):

[0014] a step of forming a complex of β₂-GPI and the cholesterolderivative immobilized on the solid phase of the present invention bybringing a specimen into contact with the solid phase (Step 1); and

[0015] a step of detecting β₂-GPI contained in the complex which hasbeen formed in Step 1 (Step 2).

[0016] The present invention is also directed to an assay method for anantibody recognizing a “complex of β₂-GPI and the derivative of thepresent invention,” characterized in that the method includes at leastthe following steps (hereinafter referred to as “assay method 2 of thepresent invention”):

[0017] a step of forming a complex of the “complex of β-GPI and thecholesterol derivative immobilized on the solid phase of the presentinvention” (hereinafter, the complex of β-GPI and the cholesterolderivative immobilized on the solid phase of the present invention isreferred to as the “β₂-GPI-cholesterol derivative complex”) and anantibody recognizing the β₂-GPI-cholesterol derivative complex bybringing β₂-GPI and a specimen into contact with the solid phase (Step1); and

[0018] a step of detecting the antibody contained in the complex whichhas been formed in Step 1 (Step 2).

[0019] The present invention is also directed to a method for detectinga disease (hereinafter referred to as the “detection method of thepresent invention”), characterized in that the method includes assayingan antibody recognizing the “complex of β₂-GPI and the derivative of thepresent invention” present in blood through the assay method 2 of thepresent invention and correlating the amount of the antibody present inblood to the disease. The disease is preferably an antiphospholipidsyndrome or thrombosis. The thrombosis is preferably arterialthrombosis.

[0020] The present invention is also directed to an assay kit forβ₂-GPI, characterized in that the kit comprises at least the following(A) and (B) (hereinafter referred to as assay kit 1 of the presentinvention):

[0021] the solid phase of the present invention (A) and

[0022] a substance binding to β₂-GPI (B).

[0023] The present invention is also directed to an assay kit for anantibody recognizing “the complex of β₂-GPI and the derivative of thepresent invention,” characterized in that the kit comprises at least thefollowing (A) and (B) (hereinafter referred to as assay kit 2 of thepresent invention):

[0024] the solid phase of the present invention (A) and

[0025] a substance binding to an antibody recognizing “the complex ofβ₂-GPI and the derivative of the present invention” (B).

[0026] The assay kit 2 of the present invention is preferably a kit fordetecting a disease. The disease is preferably an antiphospholipidsyndrome or thrombosis. The thrombosis is preferably arterialthrombosis.

[0027] The present invention will next be described in detail. First,abbreviations used in the specification are listed below.

[0028] aPL: Antiphospholipid antibody

[0029] aCL: Anticardiolipin antibody

[0030] APS: Antiphospholipid syndrome

[0031] β₂-GPI: β₂-Glycoprotein I

[0032] CL: Cardiolipin

[0033] LDL: Low-density lipoprotein

[0034] oxLDL: Oxidized LDL

[0035] Cu²⁺-oxLDL: oxLDL formed through oxidation by CuSO₄

[0036] oxLig-1: Compound represented by formula (2),9-oxo-9-(7-ketocholest-5-en-3β-yloxy)nonanoic acid, also called(7-ketocholesteryl-9-carboxynonanoate)

[0037] oxLig-2: Compound represented by formula (7),4,12-dioxo-12-(7-ketocholest-5-en-3β-yloxy)dodecanoic acid

[0038] 13-COOH-7KC: Compound represented by formula (3),13-oxo-13-(7-ketocholest-5-en-3β-yloxy)tridecanoic acid

[0039] PS: Phosphatidylserine

[0040] SLE: Systemic lupus erythematosus

[0041] TLC: Thin-layer chromatography

[0042] HPLC: High-performance liquid chromatography

[0043] Chol: Cholesterol

[0044] DOPC: Dioleoylphosphatidylcholine

[0045] [³H]DPPC: L-3-phosphatidyl[N-methyl-³H]choline, 1,2-dipalmitoyl

[0046] DPPS: Dipalmitoylphosphatidylserine

[0047] PAPC: 1-Palmitoyl-2-arachidonoyl-phosphatidylcholine

[0048] In the present specification, numerals enclosed by “[ ]” refer inprinciple to mentioned document Nos. of the below-mentioned references.

<1> Derivative of the Present Invention

[0049] The derivative of the present invention is a cholesterolderivative represented by the following formula (1):

[0050] wherein R represents a C3-C23 saturated or unsaturated aliphatichydrocarbon residue having an optional substituent. Among thehydrocarbon residues, a C5-C20 hydrocarbon residue is preferred, aC8-C15 residue is more preferred, and a C9-C13 residue is particularlypreferred. To the cholesterol backbone, —OH, —CHO, —COOH, —OOH, or anepoxy group may be added. The aforementioned R preferably has one ormore substituents selected from the group consisting of —COOH, —OH,—CHO, an oxo group, and an epoxy group.

[0051] R is preferably a C3-C23 saturated or unsaturated fatty acidresidue which may have one or more oxo groups. Among the fatty acidresidues, a C5-C20 fatty acid residue is preferred, a C8-C15 residue ismore preferred, and a C9-C13 residue is particularly preferred.

[0052] R is preferably a group represented by HOOC—R′— (wherein R′represents a C2-C22 saturated or unsaturated aliphatic hydrocarbonresidue which may have one or more oxo groups). Among the groupsrepresented by HOOC—R′—, a C4-C19 group is preferred, a C7-C14 group ismore preferred, and a C8-C12 group is particularly preferred.

[0053] R′ is preferably a linear-chain aliphatic hydrocarbon residue,with a linear-chain aliphatic hydrocarbon residue having one oxo groupbeing more preferred. The preferred number of carbon atoms of thealiphatic hydrocarbon residue is the same as described above.

[0054] Specific examples of the derivative of the present inventioninclude the cholesterol derivatives represented by the followingformulas (2) to (7), respectively.

[0055] In the present specification, the substance represented byformula (3) is referred to as “13-COOH-7KC.”

[0056] In the present specification, the substance represented byformula (7) is referred to as “oxLig-2.”

[0057] The derivative of the present invention can be produced byisolating the same from oxLDL through the method described in thebelow-mentioned example. Alternatively, the derivative can be chemicallysynthesized through the method described in the below-mentioned example.

[0058] The synthesized or isolated derivative of the present inventioncan be identified through analyses such as NMR (¹H-NMR, ¹³C-NMR)analysis, mass spectrometry, and analysis of binding performance toβ₂-GPI.

<2> Solid Phase of the Present Invention

[0059] The solid phase of the present invention is a solid phase onwhich the derivative of the present invention has been immobilized.

[0060] No particular limitation is imposed on the solid phase employedfor immobilization of the derivative of the present invention thereon,so long as the solid phase can immobilize the derivative of the presentinvention thereon and is insoluble in water, a specimen, and a reactionmixture to be assayed. Examples of the form of the solid phase includeplates (e.g., wells of a microplate), tube, beads, membrane, and gel.Examples of the solid phase material include polystyrene, polypropylene,nylon, and polyacrylamide.

[0061] Of these, a plate made of polystyrene is preferred.

[0062] In order to immobilize the derivative of the present inventiononto the solid phase, a general immobilization method for lipid; e.g.,the physical adsorption method or the covalent bond method, can beemployed.

[0063] Of these, the physical adsorption method is preferred, since themethod can be carried out in a simple manner and is often employed inthe field.

[0064] In one specific mode of the physical adsorption method, thederivative of the present invention is dissolved in a solvent, such asethanol, methanol, or a mixture of methanol and chloroform; the solutionis brought into contact with the solid phase (e.g., a microplate); andthe solvent is evaporated, thereby adsorbing the derivative of thepresent invention on the solid phase.

[0065] The surface of the solid phase on which the derivative of thepresent invention has been immobilized may include a surface portion onwhich the derivative has not yet been immobilized. If β₂-GPI or anothermolecular species contained in a specimen is attached on such a surfaceportion, accurate assay results may fail to be obtained. Thus, theportion on which the derivative of the present invention has not beenimmobilized is preferably covered with a blocking substance added priorto contact between a specimen and the solid phase. Examples of theblocking substance include serum albumin, casein, skim milk, andgelatin, and commercial blocking substance products can also be used.

[0066] In one specific blocking method, a blocking substance (e.g.,serum albumin, casein, skim milk, or gelatin) is added to the solidphase, and the solid phase is stored at about 37° C. for 30 minutes to 2hours or at ambient temperature (15 to 25° C.) for 1 to 2 hours.

<3> Assay Method of the Present Invention

[0067] The assay method 1 of the present invention is an assay methodfor β₂-GPI, characterized in that the method includes at least thefollowing steps:

[0068] a step of forming a complex of β₂-GPI and the cholesterolderivative immobilized on the solid phase of the present invention bybringing a specimen into contact with the solid phase (Step 1); and

[0069] a step of detecting β₂-GPI contained in the complex which hasbeen formed in Step 1 (Step 2).

[0070] The steps will be described individually.

[0071] Step 1:

[0072] The description set forth regarding the solid phase of thepresent invention is also applicable herein.

[0073] No particular limitation is imposed on the specimen which is tobe brought into contact with the solid phase of the present invention,so long as the specimen contains or may contain β₂-GPI, which is anassay target. Purification of the specimen in terms of β₂-GPI isoptional, and may not be performed. Specific examples of the specimeninclude blood, serum, and plasma. No particular limitation is imposed onthe method of bringing the specimen into contact with the solid phase ofthe present invention, so long as the molecules of the derivative of thepresent invention immobilized on the solid phase are brought intocontact with the β₂-GPI molecules contained in the specimen.Specifically, the specimen may be added to the solid phase of thepresent invention so as to attain contact therebetween, or the solidphase of the present invention may be added to the specimen so as toattain contact therebetween.

[0074] After contact between the specimen and the solid phase has beenattained, the system is preferably allowed to react for about one hourat, for example, 0 to 45° C., preferably 4 to 37° C., so as tosufficiently bind β₂-GPI contained in the specimen to the derivative ofthe present invention immobilized on the solid phase. After completionof reaction, preferably, the solid phase and the liquid phase aresufficiently separated from each other. Alternatively, washing of thesolid phase is preferably carried out. For example, the surface of thesolid phase is washed with a wash liquid, thereby removing non-specificadsorbed matter and non-reacted components present in the specimen.

[0075] Preferred examples of the wash liquid include buffers (e.g.,phosphate buffer, PBS, and Tris-HCl buffer) to which a non-ionicsurfactant such as a Tween series surfactant has been added.

[0076] Through contact between the specimen and the solid phase of thepresent invention, β₂-GPI contained in the specimen and the derivativeof the present invention immobilized on the solid phase form a complex,whereby β₂-GPI contained in the specimen is fixed to the solid phase bythe mediation of the derivative of the present invention.

[0077] Step 2:

[0078] No particular limitation is imposed on the detection method forβ₂-GPI included in the complex formed in Step 1. However, a substancewhich binds to β₂-GPI is preferably used.

[0079] Examples of the substance which binds to β₂-GPI include anantibody recognizing β₂-GPI. The antibody used herein may be amonoclonal antibody or a polyclonal antibody, and is appropriatelyselected in accordance with the purpose of the β₂-GPI assay, requiredprecision and sensitivity, etc. In general, when a monoclonal antibody,particularly a monoclonal antibody specifically recognizing β₂-GPI, isused, noise caused by substances other than β₂-GPI can be reduced, andhigher precision and sensitivity can be attained as compared with thecase where a polyclonal antibody is used.

[0080] The antibody against β₂-GPI can be produced through a routinepreparation method for a polyclonal antibody or a monoclonal antibodymaking use of β₂-GPI as an antigen. Alternatively, a known antibodyrecognizing β₂-GPI can also be used. Examples of such known monoclonalantibodies include EY2C9 (IgM) [36], WB-CAL-1 (IgG2a, κ) [37], andCof-22 (IgG1, κ) [38]. Characteristics of these antibodies will bedescribed in detail in the below-mentioned present example.

[0081] Such a “substance which binds to β₂-GPI” is preferably labeledwith a labeling substance, from the viewpoint of easy detection.

[0082] Even when the “substance which binds to β₂-GPI” itself is notlabeled with a labeling substance, a labeled substance which binds tothe “substance which binds to β₂-GPI” may also be used.

[0083] No particular limitation is imposed on the labeling substancesemployed for the labeling, so long as the substances can label typicalproteins. Examples include enzymes (e.g., peroxidase, alkalainephosphatase, β-galactosidase, luciferase, and acetylcholine esterase),fluorescent dyes (e.g., fluorescein isothiocyanate (FITC)), chemicalfluorescent substances (e.g., luminol), biotin, and avidin (includingstreptavidin). The labeling method is appropriately determined fromknown labeling methods suited for the labeling substance; e.g., theglutaraldehyde method, the periodate cross-linking method, the maleimidecross-linking method, the carbodiimide method, and the activated estermethod (see Chemistry of Proteins (part 2), published by Tokyo KagakuDojin, 1987). For example, when biotin is used as a labeling substance,a method employing a biotin hydrazide derivative (see Avidin-BiotinChemistry: A Handbook, p. 57-63, published by PIERCE CHEMICAL COMPANY,1994) is appropriately employed. When fluorescein isothiocyanate isused, a method disclosed in Japanese Patent Publication (kokoku) No.63-17843 or a similar method is appropriately employed.

[0084] In the case where an antibody (not labeled) against β₂-GPI isemployed as the substance which binds to β₂-GPI, another antigen(labeled) which binds to the corresponding antibody (immunoglobulin) canbe employed as a secondary antibody. More specifically, when EY2C9 isused, an anti-human-IgM antibody which has been labeled with a labelingsubstance can be used, whereas when WB-CAL-1 or Cof-22 is used, ananti-mouse IgG antibody labeled with a labelling substance can be used.Examples of the labeling substance include horseradish peroxidase (HRP).Commercial products of such a secondary antibody can be used.

[0085] By detecting a labeling substance which has been bound to β₂-GPIby the mediation of the “substance which binds to β₂-GPI,” β₂-GPIincluded in the complex formed in Step 1 can be detected.

[0086] The detection method can be appropriately determined by a personskilled in the art, in accordance with the labeling substance used. Forexample, when a peroxidase is employed as a labeling substance, hydrogenperoxide and a coloring substrate such as tetramethylbenzidine servingas a substrate for the enzyme are added to the enzyme reaction system,and the degree of coloring of the product can be detected by measuringthe change in absorbance. When a fluorescent substance orchemiluminescent substance is used, detection can be performed bymeasuring fluorescence or luminescence provided from the solution aftercompletion of reaction.

[0087] In the present specification, the term “detection” refers notonly to qualitative detection (i.e., detection to check the presence orabsence of the substance to be detected), but also to quantitativedetection (i.e., detection to determine the amount (concentration) ofthe substance to be detected). The same convention is also applied tothe term “assay” in the present specification.

[0088] When quantitative detection (assay) is performed, a calibrationcurve representing the relationship between the β₂-GPI concentration andcertain detected values (e.g., absorbance) of the standard substance isprepared in advance by use of a β₂-GPI standard solution of a knownconcentration. Then, a specimen having an unknown concentration of thesubstance to be detected is subjected to measurement, and theconcentration can be determined from the detected values by use of thecalibration curve.

[0089] The assay method 2 of the present invention can be provided byfurther modifying the assay method 1 of the present invention. In theassay method 2, a specimen is brought into contact with the “complex ofβ₂-GPI and the derivative of the present invention immobilized on thesolid phase” formed in Step 1 of the assay method 1 of the presentinvention, and the antibody recognizing the “complex of β₂-GPI and thederivative of the present invention” contained in the specimen isassayed.

[0090] Specifically, the assay method 2 is an assay method for anantibody recognizing the “complex of β₂-GPI and the derivative of thepresent invention,” characterized in that the method includes at leastthe following steps:

[0091] a step of forming a complex of the “β₂-GPI-cholesterol derivativecomplex” and an antibody recognizing the “β₂-GPI-cholesterol derivativecomplex” by bringing β₂-GPI and a specimen into contact with the solidphase (Step 1); and

[0092] a step of detecting the antibody contained in the complex whichhas been formed in Step 1 (Step 2).

[0093] The steps will be described individually.

[0094] Step 1:

[0095] The aforementioned description regarding the solid phase of thepresent invention is also applicable herein.

[0096] The β₂-GPI to be brought into contact with the solid phase of thepresent invention may or may not be completely purified, and the degreeof purification may be appropriately determined by a person skilled inthe art in accordance with factors such as the desired sensitivity.However, purified β₂-GPI is preferably used. Purification of β₂-GPI canbe performed through, for example, the method described in thebelow-mentioned example.

[0097] The same description as provided in relation to the assay method1 of the present invention is also applied to the specimen to be broughtinto contact with the solid phase of the present invention. The samecontact method as employed in the assay method 1 of the presentinvention is applied to the contact method for bringing β₂-GPI and thespecimen into contact with the solid phase of the present invention. Noparticular limitation is imposed on the contact method, so long as themethod ensures the chance to attain contact between β₂-GPI molecules andthe molecules of the derivative of the present invention immobilized onthe solid phase of the present invention and the chance to attaincontact between the “complex of β₂-GPI and the derivative of the presentinvention” and the antibody molecules present in the specimen andrecognizing the complex.

[0098] Similar to the assay method 1 of the present invention, aftercontact between the complex and the antibody has been attained, thesystem is preferably allowed to react for about one hour at, forexample, 0 to 45° C., preferably 4 to 37° C., so as to sufficiently bindβ₂-GPI to the derivative of the present invention immobilized on thesolid phase and also to sufficiently bind the antibody present in thespecimen to the “complex of β₂-GPI and the derivative of the presentinvention.” The same description as provided in relation to washing orother operation after reaction performed in the assay method 1 of thepresent invention is also applied herein.

[0099] By bringing the specimen and β₂-GPI into contact with the solidphase of the present invention, β₂-GPI and the derivative of the presentinvention immobilized on the solid phase form a complex, and theantibody recognizing the “β₂-GPI-cholesterol derivative complex”contained in the specimen binds to the complex, whereby the antibodypresent in the specimen is fixed to the solid phase by the mediation ofthe complex of β₂-GPI and the derivative of the present invention.

[0100] Step 2:

[0101] No particular limitation is imposed on the detection method forthe antibody included in the complex formed in Step 1. However,preferably, a substance which binds to the antibody present in aspecimen is employed. Examples of the substance which binds to theantibody include an antibody recognizing an antibody (immunoglobulin)present in the specimen. For example, in the case of assay of anantibody present in human serum (autoantibody), an anti-human-IgGantibody can be employed as a secondary antibody.

[0102] The description provided with respect to the substance whichbinds to the antibody (e.g., secondary antibody) employed in the assaymethod 1 of the present invention applies herein mutatis mutandis.Similar to the assay method 1 of the present invention, the substancewhich binds to the antibody is preferably labeled with a labelingsubstance. The descriptions provided with respect to the assay method 1of the present invention regarding the labeling substance employed as alabel, the labeling method, and the detection method for the labelingsubstance apply herein mutatis mutandis.

<4> Detection Method of the Present Invention

[0103] The detection method of the present invention is a method fordetecting a disease, characterized in that the method includes assayingan antibody present in blood and recognizing the “complex of β₂-GPI andthe derivative of the present invention” through the assay method 2 ofthe present invention and correlating the amount of the antibody presentin blood to the disease.

[0104] In the detection method of the present invention, firstly, anantibody recognizing the “complex of β₂-GPI and the derivative of thepresent invention” and present in blood is assayed through the assaymethod 2 of the present invention. The aforementioned assay method 2 ofthe present invention is also applied herein. Although the specimen usedherein is a “blood,” the specimen is not necessarily a whole blood, andother blood samples may be used so long as the samples reflect theamount of the antibody present in blood. Specifically, there may also beused a plasma or a serum derived from the blood, a diluted productthereof, or a product thereof modified within a degree so as not toaffect the antibody present in the sample.

[0105] In the detection method of the present invention, secondly, adisease is detected by correlating, to the disease, the amount(concentration) of the antibody present in such a specimen. The “amountof the antibody” may be the aforementioned antibody level (an actuallymeasured value) obtained by use of the calibration curve which has beenprepared on the basis of the relationship between the concentration ofthe standard antibody product and certain detected values of the labeledsubstance. Alternatively, the “amount of the antibody” may be a ratio (arelative value) of the antibody level to the amount of antibody in bloodof a healthy subject (a human not suffering the disease to be detected)which ratio is obtained without using the calibration curve.

[0106] The aforementioned antibody level increases in the presence of acertain disease. Therefore, when the blood antibody level is lower thanthat of a healthy subject, such a low level can be correlated to thestate of “suffering the disease” or the state of “highly likelysuffering the disease.” When the blood antibody level is equal to thatof a healthy subject, the level can be correlated to the state of “notsuffering the disease” or the state of “less likely suffering thedisease.”

[0107] In addition to check whether a subject suffers a certain diseaseor not, the detection method of the present invention includes detectionof the degree of suffering the disease. For example, in a person whoperiodically undergoes an assay of the blood antibody level, when theantibody level tends to increase, the tendency can be correlated to thestate of “progress of the disease” or the state of “highly likelyprogress of the disease.” In contrast, when the assayed antibody leveltends to decrease, the tendency can be correlated to the state of“amelioration of the disease” or the state of “highly likelyamelioration of the disease.” When the assayed antibody level isconstant, the constant amount can be correlated to the state of “nochange in sickness (healthiness)” or the state of “highly likely nochange in sickness (healthiness).”

[0108] The “disease” is preferably an antiphospholipid syndrome orthrombosis. The thrombosis is preferably arterial thrombosis.

<5> Kit of the Present Invention

[0109] The kit 1 of the present invention is an assay kit for β₂-GPI,characterized in that the kit comprises at least the following (A) and(B):

[0110] the solid phase of the present invention (A) and

[0111] a substance binding β₂-GPI (B).

[0112] The kit 2 of the present invention is an assay kit for anantibody recognizing “the complex of β₂-GPI and the derivative of thepresent invention,” characterized in that the kit comprises at least thefollowing (A) and (B):

[0113] the solid phase of the present invention (A) and

[0114] a substance binding to an antibody recognizing “the complex ofβ₂-GPI and the derivative of the present invention” (B).

[0115] The aforementioned description regarding the solid phase of thepresent invention is also applicable herein. The descriptions providedwith respect to the assay methods 1 and 2 of the present inventionapply, mutatis mutandis, to the substance binding to β₂-GPI and to thesubstance binding to an antibody recognizing “the complex of β₂-GPI andthe derivative of the present invention.” No particular limitation isimposed on the constitution of the assay kits 1 and 2 of the presentinvention, so long as each kit contains the aforementioned (A) and (B).The kits may further include, as an additional component, for example, aspecies of known concentration serving as a standard for preparing acalibration curve (β₂-GPI (kit 1) and an antibody recognizing “thecomplex of β₂-GPI and the derivative of the present invention” (kit 2));a reagent for detecting a labeling substance; a reagent which labels thesubstance binding to β₂-GPI or which labels the substance binding to theantibody recognizing “the complex of β₂-GPI and the derivative of thepresent invention”; or a labeled substance thereof. In addition to theseadditional components, the kits may further contain, for example, ablocking substance, the aforementioned washing liquid, aspecimen-diluting agent, or an enzymatic reaction-stopping agent.

[0116] These kit components may be placed separately in individualcontainers and can be stored until use thereof as a kit which can beused in accordance with the assay method of the present invention.

[0117] The assay of β₂-GPI by use of the kit 1 of the present inventioncan be performed in accordance with the assay method 1 of the presentinvention, and the assay of the antibody recognizing “the complex ofβ₂-GPI and the derivative of the present invention” by use of the kit 2of the present invention can be performed in accordance with the assaymethod 2 of the present invention.

[0118] The kit 2 of the present invention is preferably a kit fordetecting a disease. The disease is preferably an antiphospholipidsyndrome or thrombosis. The thrombosis is preferably arterialthrombosis. In this case, the detection of a disease can be performed inaccordance with the detection method of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0119]FIG. 1. Molecular interactions among β₂-GPI, LDLs, lipoproteins(PLs), and anti-β₂-GPI autoantibody, detected by an optical biosensor.

[0120] A: LDL (open square) or oxLDL (closed square) binding to solidphase β₂-GPI;

[0121] B: LDL (open square) or oxLDL (closed square) binding to solidphase WB-CAL-1 in the presence of β₂-GPI;

[0122] C: DOPE (open circle) or CL (closed circle) binding to solidphase β₂-GPI; and

[0123] D: DOPE (open circle) or CL (closed circle) binding to solidphase WB-CAL-1 in the presence of β₂-GPI.

[0124]FIG. 2. Binding of β₂-GPI and anti-β₂-GPI autoantibodies to plasmaLDLs or their lipid extracts:

[0125] A: Plasma LDLs, β₂-GPI, and mouse anti-human β₂-GPI monoclonalantibody (Cof-22) were sequentially incubated in a plate coated with Fabfragment of anti-apoB100 monoclonal antibody (1D2). Binding was detectedusing HRP-labeled anti-mouse IgG.

[0126] B to D: Lipid extracts from LDLs were applied to a plate. β₂-GPIbinding was detected using Cof-22 and using HRP-labeled anti-mouse IgG(B). Subsequent binding of WB-CAL-1 (C) and EY2C9 (D) were detectedusing HRP-labeled anti-mouse IgG (C), and with HRP-labeled anti-humanIgM (D), respectively. Open columns: without β₂-GPI, closed columns:with β₂-GPI (C, D). Data are indicated as the mean±SD of triplicatesamples.

[0127]FIG. 3. Thin-layer chromatography (TLC) and ligand blot of lipidextracts from LDLs.

[0128] Lipids extracted from LDLs were spotted on silica gel plates anddeveloped in solvent A.

[0129] A: Staining with I₂ vapor, molybdenum blue, orcin/sulfuric acid,and sulfuric acid/acetic acid as indicated in the figure.

[0130] B: Ligand blot was performed with β₂-GPI and anti-β₂-GPIantibodies. The region marked with an asterisk was scraped off andsubjected to further purification. Glu-Cer represents glucosylceramide,and Gal-Cer represents galactosylceramide.

[0131] C: Ligand blot of the scraped lipids (Band-1 and Band-2) wasperformed by sequential treatment with β₂-GPI and anti-β₂-GPI antibodies(2-step)

[0132] D: Ligand blot of the eluate was performed by co-incubation ofβ₂-GPI and anti-β₂-GPI antibodies (1-step).

[0133]FIG. 4. Elution profiles of Band-1 by reversed phase highperformance liquid chromatography (HPLC).

[0134] Scraped Band-1 was eluted on Sephacil-Peptide column and detectedat 210 nm (A) and 234 nm (B). Data of ligand blot analysis on eluateusing EY2C9 are also shown in (C).

[0135]FIG. 5. LC/MS of purified oxLig-1 and its methylated compound.

[0136] A: HPLC profiles of oxLig-1 (upper) and methylated oxLig-1(lower) at 234 nm.

[0137] B: A positive ionization mass spectrum of 7-ketocholesterol.

[0138] C: A negative ionization mass spectrum of oxLig-1.

[0139] D: A positive ionization mass spectrum of oxLig-1.

[0140] E: A negative ionization mass spectrum of methylated oxLig-1.

[0141] F: A positive ionization mass spectrum of methylated oxLig-1.

[0142]FIG. 6. Synthesis of oxLig-1(9-oxo-9-(7-ketocholest-5-en-30β-yloxy)nonanoic acid) and itsmethylation.

[0143]FIG. 7. Nuclear magnetic resonance (NMR) spectrum of synthesizedoxLig-1 and its methylated compound. 300 MHz ¹H-NMR spectra ofsynthesized oxLig-1 (A) and of methylated oxLig-1 (C). 75.3 MHz ¹³C-NMRspectra of synthesized oxLig-1 (B) and of methylated synthesized oxLig-1(D).

[0144]FIG. 8. TLC and ligand blot analysis on synthesized oxLig-1.

[0145] A: oxLig-1 and synthesized oxLig-1 was spotted on a TLC plate,developed with solvent A, and detected with I₂-vapor.

[0146] B: Ligand blot of synthesized oxLig-1 was performed withanti-β₂-GPI antibodies in the presence (+) or absence (−) of β₂-GPI.

[0147]FIG. 9. LC/MS of synthesized oxLig-1 and its methylated compound.

[0148] A: LC chromatograms of synthesized oxLig-1 at 210 nm and at 234nm.

[0149] B: A positive ionization mass spectrum of synthesized oxLig-1.

[0150] C: A negative ionization mass spectrum of synthesized oxLig-1.

[0151] D: LC chromatograms of methylated synthesized oxLig-1 at 210 nmand at 234 nm.

[0152] E: A positive ionization mass spectrum of methylated oxLig-1.

[0153] F: A negative ionization mass spectra of methylated synthesizedoxLig-1.

[0154]FIG. 10. Effect of phosphatidylserine (PS) or oxLig-1 content onthe binding of liposomes to macrophages. A monolayer of J774.A1 cellswas incubated for 2 hours at 4° C. with Celgrosser-P medium containing³H-labeled liposomes (50 nmol lipid/well). Open circle representsPS-liposomes; closed circle represents PS-Chol-liposomes; open squarerepresents oxLig-1-liposomes; and closed square representsoxLig-1-Chol-liposomes. Data are indicated as the mean±SD of triplicatesamples.

[0155]FIG. 11. β₂-GPI and anti-β₂-GPI monoclonal antibody-dependentbinding of ligand-containing liposomes to macrophage. A monolayer ofJ774.A1 cells was incubated for 2 hours at 4° C. with Celgrosser-Pmedium containing ³H-labeled liposomes (50 nmol lipid/well) andWB-CAL-1, in the presence or absence of β₂-GPI (200 μg/ml)

[0156] A: Binding of PS-liposomes (PS: 50 mol %,) to J774.A1. cells inthe presence (closed square) or absence (open square) of β₂-GPI (200μg/ml)

[0157] B: Binding of oxLig-1-liposomes (oxLig-1: 40 mol %) to J774.A1cells in the presence (black bar) or absence (white bar) of β₂-GPI (200μg/ml)

[0158] C: A monolayer of J774.A1 cells was incubated for 2 hours at 4°C. with Celgrosser-P medium containing ³H-labeled synthesizedoxLig-1-liposomes (50 nmol lipid/well) and WB-CAL-1, in the presence orabsence of β₂-GPI (200 μg/ml).

[0159] Binding of synthesized oxLig-1-liposomes in the absence of β₂-GPIand WB-CAL-1 (open circle), binding of synthesized oxLig-1-liposomes inthe presence of β₂-GPI (open square), binding of synthesizedoxLig-1-liposomes in the presence of WB-CAL-1 (closed square), andbinding of synthesized oxLig-1-liposomes in the presence of both β₂-GPIand WB-CAL-1 (closed circle) Data are indicated as the mean±SD oftriplicate samples.

[0160]FIG. 12. Autoantibodies present in APS serum samples against solidphase β₂-GPI-oxLig-1 complex. Serum samples were obtained from healthysubjects (n=24) and APS patients with episodes of thrombosis (n=52).

[0161] A: Antibody values in individual serum samples

[0162] B: Relationship between anti-β₂-GPI-oxLig-1 antibody values andβ₂-GPI-dependent aCL values

[0163] C: Relationship between anti-β₂-GPI-oxLig-1 antibody values andanti-β₂-GPI antibody values.

[0164]FIG. 13. Relationship between anti-β₂-GPI-oxLig-1 antibody valuesand antibody values of the β₂-GPI-dependent aCL (anti-β₂-GPI-CLantibody) in ELISA, and relationship between anti-β₂-GPI-oxLig-1antibody values and anti-β₂-GPI antibody values in ELISA. Plasma samplesof 133 APS and/or SLE patients (87 APS patients and 47 SLE onlypatients) were assayed through the method described in relation tomaterials and methods.

[0165]FIG. 14. Relationship between antibody values and clinicalepisodes. Plots of anti-β₂-GPI-oxLig-1 antibody values of APS and/or SLEpatients (without clinical episodes (open circles), with clinicalepisodes (gray circles)). The values of p represent Mann-Whitney U-testresults. The dashed lines represent a cut-off value (the level exceedingthe averaged (healthy control) antibody value by 3×SD).

[0166]FIG. 15. TLC and Ligand blot profiles of oxLig-2, Me-oxLig-2,13-COOH-7KC, and Me-13-COOH-7KC.

[0167]FIG. 16. Elution profiles of Band-2 obtained by reversed phasehigh performance liquid chromatography (HPLC).

[0168] Scraped Band-2 was eluted on Sephacil-Peptide column and detectedat 210 nm (A) and 234 nm (B). Data of ligand blot analysis on eluateusing EY2C9 are also shown in (B). Fraction 14 was purified againthrough HPLC under the same conditions so as to confirm the purity (Cand D).

[0169]FIG. 17. LC/MS of purified or synthesized β₂-GPI ligands. Positiveionization mass spectra of oxLig-1 (A), oxLig-2 (C), and 13-COOH-7KC (E)(left column) and negative ionization mass spectra of oxLig-1 (B),oxLig-2 (D), and 13-COOH-7KC (F) (right column).

[0170]FIG. 18. Structures of cholesteryl esters serving as β₂-GPIligands.

[0171] Structures of cholesteryl linoleate (A), oxLig-1 (B), oxLig-2(C), and 13-COOH-7KC (E). Schemes of fragmentation possibly occurring inmass spectroscopy are specified by means of arrows. Scission at eacharrow position will result in formation of the corresponding fragmention “D.”

[0172]FIG. 19. Direct binding of ligand-containing liposomes tomacrophage.

[0173] A monolayer of J774.A1 cells was incubated for 2 hours at 4° C.with Celgrosser-P medium containing [³H]-labeled liposomes (containing apredetermined ligand, 50 nmol lipid/well). DPPS-containing liposomes(open squares), oxLig-1-containing liposomes (closed squares),oxLig-2-containing liposomes (closed circles), and13-COOH-7KC-containing liposomes (closed triangles). Data are indicatedas mean±SD of triplicate samples.

[0174]FIG. 20. β₂-GPI and anti-β₂-GPI antibody-dependent binding ofligand-containing liposomes to macrophage.

[0175] A monolayer of J774.A1 cells was incubated for 2 hours at 4° C.with Celgrosser-P medium containing [³H]-labeled liposomes (containing apredetermined ligand (30 mmol %), 50 nmol lipid/well) and in thepresence (black bars) or absence (white bars) of β₂-GPI (200 μg/mL) andWB-CAL-1 (200 μg/mL).

[0176] Chart A: Cholesteryl linoleate-containing liposomes; Chart B:oxLig-1-containing liposomes, Chart C: oxLig-2-containing liposomes; andChart D: 13-COOH-7KC-containing liposomes. In Charts C and D, comparisonwas made with methylated ligand-containing liposomes. Data are indicatedas mean±SD of triplicate samples.

BEST MODE FOR CARRYING OUT THE INVENTION

[0177] The present invention will next be described in more detail byway of example, which should not be construed as limiting the inventionthereto.

<1> Materials and Methods

[0178] Firstly, materials and methods employed in the present examplewill be described.

[0179] (1) Chemicals

[0180] L-α-Dipalmitoylphosphatidylserine (DPPS), CL, Chol, and7-ketocholesterol (5-cholesten-3β-ol-7-one) were obtained from SigmaChemical Co. PAPC and DOPC from Avanti Polar Lipids Inc. [³H]DPPC (80Ci/mmol) from Amersham-Pharmacia Biotech. Other chemicals were obtainedfrom commercial sources and of reagent grade quality.

[0181] (2) Purification of Human β₂-GPI

[0182] β₂-GPI was purified from normal human plasma as described in [35]with slight modification. Pooled plasma from healthy subjects werechromatographed on a heparin-Sepharose column (or CL-polyacrylamide gelcolumn), on a DEAE-cellulose column, and on an anti-β₂-GPI affinitycolumn. To remove any contamination by IgGs, the β₂-GPI-containingfraction was further passed through a protein A-Sepharose column. Thefinal β₂-GPI fraction was delipidated by extensive washing withn-butanol.

[0183] (3) Anti-β₂-GPI positive serum samples were obtained from APSpatients with episodes of arterial thrombosis. To eliminate endogenousβ₂-GPI in some experiments, serum samples were passed through aheparin-Sepharose column. The effluent was dialyzed against PBS and wasused for ELISAs.

[0184] (4) Monoclonal Antibodies (mAbs)

[0185] EY2C9 (IgM): A human monoclonal anti- ₂-GPI autoantibody,established from peripheral blood lymphocytes from an APS patient [36].

[0186] WB-CAL-1 (IgG2a, κ): A mouse monoclonal anti-β₂-GPI autoantibody,derived from an (NZW×BXSB) F1 mouse [37].

[0187] Cof-22 (IgG1, κ): A monoclonal anti-human β₂-GPI antibody,established from BALB/c mice immunized with human β₂-GPI [38].

[0188] Both EY2C9 and WB-CAL-1 bind either to a complex of β₂-GPI and CLand to β₂-GPI adsorbed on an oxygenated polystyrene plates. In contrast,Cof-22 is specific for human β₂-GPI and recognizes its native structure.

[0189] (5) Preparation of oxLDL and Lipid Extraction

[0190] Plasma LDL (1.019<d<1.063 g/mL) was isolated byultracentrifugation from fresh normal human plasma, as described in[39]. LDL (100 μg protein/mL) was oxidized by incubating with 5 μM CuSO₄(PBS solution) for 8 hours at 37° C. To stop the oxidation, 1 mM EDTAwas added. The oxidized sample was extensively dialyzed against PBScontaining 1 mM EDTA.

[0191] Protein concentration was determined using BCA protein assayreagent (product of Pierce Chemical Co.). An aliquot of a sample wastaken to determine thiobarbituric acid reactive substance (TBARS) valueserving as an index of an extent of oxidation [40], and for use inagarose gel electrophoresis. A lipid fraction was extracted from LDLsaccording to the method described in [41].

[0192] (6) Assay for Molecular Interaction

[0193] Real-time molecular analysis was performed using an opticalbiosensor, IAsys (product of Affinity Sensors).

[0194] Binding of β₂-GPI to LDLs or liposomes: Biotinyl-β₂-GPI wasimmobilized on a biotinyl-cuvette via streptavidin. LDLs or liposomes atvarious concentrations were placed in the cuvette. Antibodies binding toLDLs or liposomes: The biotinyl-WB-CAL-1 was immobilized on the cuvette.LDLs or liposomes were added at various concentrations in the presence(10 μg/mL) or absence of β₂-GPI.

[0195] (7) ELISA for Detecting Binding of β₂-GPI to anti-β₂-GPI Antibody

[0196] Binding to LDLs: A microtiter plate (Immulon 2HB, DynexTechnologies Inc.) was coated with 50 μL of F(ab′)2 of 1D2 (anti-apoB100 antibody; 10 μg/mL, product of Yamasa Corp.) by incubation overnightat 4° C. After blocking with PBS containing 1% skim milk, the plate wasincubated with LDLs for one hour. The wells were incubated sequentiallywith β₂-GPI (15 μg/mL), Cof-22, and horseradish peroxidase (HRP)-labeledanti-mouse IgG, each for one hour. The color was developed with H₂O₂ ando-phenylenediamine, and OD was measured at 490 nm.

[0197] Binding to extracted lipids: A microtiter plate (Immulon 1B,Dynex Technologies Inc.) was coated with lipids extracted from LDLs (50μg/mL, 50 μL/well) by ethanol evaporation. The wells were blocked withPBS containing 1% BSA for one hour and the wells were incubated with 30μg/mL of β₂-GPI for one hour. Then, β₂-GPI binding was detected usingCof-22 and HRP-labeled anti-mouse IgG antibodies. Alternatively, ananti-β₂-GPI autoantibody (Cof-22, WB-CAL-1, or EY2C9) was diluted with a0.3% BSA-containing PBS, and the diluted antibody was added to thecoated plate (1.0 μg/mL, 100 μL/well) and incubated with β₂-GPI (15μg/mL) for one hour. The binding of the antibody was detected by anHRP-labeled anti-mouse IgG /or an anti-human IgM. The color wasdeveloped with H₂O₂ and o-phenylenediamine, and OD was measured at 490nm. Between these steps, extensive washing were done using PBScontaining 0.05% Tween 20.

[0198] (8) Thin Layer Chromatography (TLC) and Ligand Blot Analysis

[0199] Extracted lipids were spotted on a Polygram silica gel plate(product of Machery-Nagel) and developed in chloroform/methanol/30%ammonia/water (120:80:10:5, v/v/v/v, hereinafter referred to as solventA). The plate was stained with 12 vapor, or with a spray of molybdenumblue, of 2N sulfuric acid containing 2% orcin, or of glacial aceticacid/sulfuric acid (19:1, v/v) (Lieberman-Burchard reaction).Alternatively, the developed plate was subjected to ligand blot withβ₂-GPI and an anti-β₂-GPI antibody. TLC plates were blocked with PBScontaining 1% bovine serum albumin (BSA) and were subsequently incubatedwith β₂-GPI, anti-β₂-GPI antibodies (Cof-22, WB-CAL-1, or EY2C9), andHRP-labeled anti-mouse IgG antibodies or anti-human IgM antibodies forone hour. In each step, plates were extensively washed with PBS. Thecolor was developed with H₂O₂ and 4-methoxy-1-naphtol (product ofAldrich). The ligand-enriched bands scraped from the TLC plate weresubjected to another TLC in chloroform/methanol (8:1, v/v) (hereinafterreferred to as solvent B). For large scale purification of the ligand,extracted lipids were loaded on a TLC silica gel 60 plate (PLC plate;product of Merck) of 2 mm thickness.

[0200] (9) HPLC

[0201] A fraction rich in a ligand (originating from oxLDL) obtained bytwo-step TLC was analyzed by reversed phase HPLC on a Sephasil PeptideC-18 5-μm column (4.6 mm×250 mm; product of Amersham-Pharmacia Biotech).The column underwent elution with a mixture ofacetonitrile/isopropanol/water (60:30:2, v/v/v, hereinafter referred toas solvent C) at a flow rate of 0.5 mL/min and fractionated everyminute. Each eluate was spotted on a TLC plate and subjected to ligandblot with β₂-GPI and EY2C9.

[0202] (10) NMR

[0203] 1H-NMR and ¹³C-NMR spectra were recorded by means of ASX-300spectrometer (Bruker).

[0204] (11) Mass Spectroscopy

[0205] Synthesized oxLig-1 was analyzed on a Shim-pack VP-ODS column(4.6 mm×150 mm) with an LC/MS-QP8000α (Shimadzu Corp.), using solvent C(LC/MS; liquid chromatography/mass spectroscopy). Positive and negativeionization mass spectra were recorded in the mass range of 50-850.

[0206] Mass spectrometry of oxLig-1, oxLig-2 purified from Band-2, andsynthesized 13-COOH-7KC, which are β₂-specific ligands, was carried outon a Shim-pack FC-ODS column (4.6 mm×30 mm) with an LC/MS-2010spectrometer (Shimadzu Corp.), using solvent F (30% acetone in methanol)and water with a linear gradation in concentration (50 to 100%).Positive and negative ionization mass spectra were taken in a mass rangeof 50 to 750.

[0207] The field desorption (FD) mass spectra of synthesized oxLig-1 andmethylated oxLig-l were recorded on JMS-SX102A and JMS AX-500spectrometers (product of JEOL), respectively.

[0208] (12) Synthesis of β₂-GPI-Specific Ligand (oxLig-1)

[0209] The ligand, oxLig-1, was first isolated from Cu²⁺-oxLDL throughtwo steps of TLC, followed by HPLC, and finally identified as9-oxo-9-(7-ketocholest-5-en-3β-yloxy)nonanoic acid(7-ketocholesteryl-9-carboxynonanoate) (FIG. 1). In the present example,the synthesized oxLig-1 was used instead of purified oxLig-1. To asolution of 7-ketocholesterol (5-cholesten-3β-ol-7-one, 50.1 mg, 0.125mmol) and azelaic acid (70.6 mg, 0.375 mmol) in acetone (4 mL) wereadded 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (95.8mg, 0.50 mmol) and 4-(dimethylamino)pyridine (30.5 mg, 0.25 mmol). Themixture was stirred at room temperature for two days, concentrated, andextracted with chloroform. The extract was successively washed with 2Mhydrochloric acid and dried over magnesium sulfate anhydrate, and thesolvent was evaporated. The residue was subjected to columnchromatography on silica gel using toluene/ethyl acetate (3:1, v/v), tothereby yield oxLig-1 (36.0 mg, yield: 50.4%).

[0210] (13) Analysis Results of Synthesized oxLig-1

[0211] (NMR);

[0212]¹H-NMR (300 MHz, CDCl₃): δ=5.71 (s, ¹H, H-6), 4.78-4.69 (m, ¹H,H-3);

[0213]¹³C-NMR (75.5 MHz, CDCl₃): δ=202.5, 179.7, 173.4, 164.5, 127.1,72.4, 55.2, 50.4, 50.2, 45.8, 43.5, 39.9, 38.7, 36.6, 36.1, 29.2, 28.9,28.4, 25.3, 25.0, 24.2, 23.2, 23.0, 19.3, 17.7, 12.4.

[0214] (Mass spectroscopy);

[0215] m/z (FD-MS): 571 [(M+H)⁺, C₃₆H₅₉O₅].

[0216] (14) Methylation of oxLig-1

[0217] To a solution of purified or synthesized oxLig-1 (7.5 mg, 0.0131mmol) in ether (2 mL), a diazomethane-ether solution was added at roomtemperature, and the mixture was stirred for 2 hours. Acetic acid wasadded to the solution while stirring. One day after addition of aceticacid, the solvent was evaporated to give methylated oxLig-1 preparation.

[0218] Hereinafter, a methylated species may be represented with aprefix “Me-.”

[0219] (15) Analysis Results of Methylated Synthesized oxLig-1

[0220] (NMR);

[0221]¹H-NMR (300 MHz, CDCl₃): d=5.71 (s, ¹H, H-6), 4.78-4.69 (m, m, ¹H,H-3), 3.67 (s, ¹H, COOCH3);

[0222]¹³C-NMR (75.5 MHz, CDCl₃): d=202.4, 174.7, 173.4, 164.4, 127.1,72.4, 55.2, 50.4, 45.8, 43.5, 39.9, 38.7, 36.6, 36.4, 36.1, 29.3, 28.4,26.7, 25.3, 24.2, 23.2, 23.0, 21.6, 19.3, 17.7, 12.4.

[0223] (Mass spectroscopy);

[0224] m/z (FD-MS): 585 [(M+H)⁺, C₃₇H₅₁O₅].

[0225] (16) Methylation of oxLig-1

[0226] Under cooling with ice, 1-methyl-3-nitro-1-nitrosomethylguanidine(0.20 g) was added to a mixture of 2M sodium hydroxide (10 mL) anddiethyl ether (10 mL). The resultant mixture was stirred for severalminutes, whereby a pale-yellow liquid was separated as an upper layer.The thus-separated liquid was employed for methylation. A diazomethanesolution (2 mL) was added dropwise to a solution (1 mL) of a lipidligand (oxLig-2 or 13-COOH-7KC) (1.0 mg) in diethyl ether at 0° C. Eachof the formed two liquids was stored overnight in a refrigerator. TLCanalysis revealed that each starting substance was completely converted.The solvent of each solution was removed through air blow, to therebyyield a methyl ester of each ligand as a white, amorphous matter.

[0227] (17) Synthesis of 13-COOH-7KC

[0228] WSC (95.8 mg, 50 mmol) and DMAP (30.5 mg, 0.25 mmol) were addedto an acetone solution (4 mL) containing 7-ketocholesterol(5-cholesten-3β-ol-7-one) (50.1 mg, 0.13 mmol) and tridecanedioic acid(brassylic acid) (61.8 mg, 0.25 mmol). The mixture was stirred at roomtemperature for two days, and the reaction mixture was concentrated andextracted with chloroform. The extract was sequentially washed with 2Mhydrochloric acid, an aqueous saturated sodium hydrogencarbonatesolution, and brine. The thus-obtained liquid was dried over magnesiumsulfate anhydrate, and the solvent was evaporated. The residue wasapplied to silica gel chromatography using toluene/ethyl acetate (3:1,v/v), to thereby yield 44 mg of 13-COOH-7KC (yield: 56.0%). NMR spectraand a mass spectrum (FD-MS) of the product were recorded in the samemanner as described above.

[0229] (NMR);

[0230]¹H-NMR (300 MHz, CDCl₃): δ=5.69 (s, ¹H, H-6), 4.80-4.67 (m, ¹H,H-3);

[0231]¹³C-NMR (75.5 MHz, CDCl₃): δ=202.2, 179.6, 173.1, 164.7, 126.9,72.3, 55.3, 50.5, 50.1, 45.3, 43.5, 40.6, 39.2, 38.6, 36.5, 36.0, 29.1,28.8, 28.3, 25.2, 24.9, 24.1, 23.1, 22.9, 19.2, 17.6, 12.3.

[0232] (Mass spectroscopy);

[0233] m/z (FD-MS): 627 [(M+H)⁺, C₄₀H₆₇O₅].

[0234] (18) Preparation of Liposomes

[0235] Liposomes were prepared through a method as described in [42],with the following lipid compositions. Lipid proportions by mol of 0,10, 25, and 50% PS-liposomes are as follows.

[0236] DOPC/DPPS/[³H]-DPPC (80 Ci/mmol)

[0237] 0%: 100/0/0.00225

[0238] 10%: 90/10/0.00225

[0239] 25%: 75/25/0.00225

[0240] 50%: 50/50/0.00225

[0241] Lipid proportions by mol of 0, 5, 12.5, and 25% PS-Chol-liposomesare as follows.

[0242] DOPC/DPPS/Chol/ [³H]-DPPC (80 Ci/mmol)

[0243] 0%: 50/0/50/0.00225

[0244] 5%: 45/5/50/0.00225

[0245] 12.5%: 37.5/12.5/50/0.00225

[0246] 25%: 25/25/50/0.00225

[0247] Lipid proportions by mol of 0, 10, 20, and 40% oxLig-1 (purifiedproduct)-liposomes are as follows.

[0248] DOPC/oxLig-1/[³H]-DPPC (80 Ci/mmol)

[0249] 5%: 100/0/0.00225

[0250] 10%: 90/10/0.00225

[0251] 20%: 80/20/0.00225

[0252] 40%: 60/40/0.00225)

[0253] Lipid proportions by mol of 0, 5, 10, 20, 30, and 40% oxLig-1(purified product)-Chol-liposomes are as follows.

[0254] DOPC/Chol/oxLig-1/[³H]-DPPC (80 Ci/mmol)

[0255] 50%: 50/50/0/0/0.00225

[0256] 5%: 50/45/5/0.00225

[0257] 10%: 50/40/10/0.00225

[0258] 20%: 50/30/20/0.00225

[0259] 30%: 50/20/30/0.00225

[0260] 40%: 50/10/40/0.00225

[0261] oxLig-1 (synthesized product)-Liposomes were prepared usingsynthesized oxLig-1 in a manner similar to preparation of oxLig-1(purified product)-liposomes. A mixture of the desired lipids inchloroform/methanol (1:1, v/v) was placed in a pare-shaped flask and thesolvent was removed in a rotary evaporator under reduced pressure. Thedried lipids were dispersed with a mixer in 0.3M glucose solution. Thenthe liposome solution was sonicated for 5 minutes at 70° C. with aprobe-type sonicator.

[0262] Liposomes of oxLig-2, 13-COOH-7KC, etc. were also prepared in asimilar manner by use of the following lipid compositions. Liposomescontaining each ligand in an amount of 0, 10, 25, 30, and 50% wereprepared by use of a DOPC/ligand/[³H]-DPPC (80 Ci/mmol). The amount of[³H]-DPPC used was 0.225%. As the ligands, cholesteryl linoleate, DPPS,oxLig-l, oxLig-2, methylated oxLig-2 (Me-oxLig-2), 13-COOH-7KC, andmethylated 13COOH-7KC (Me-13COOH-7KC) were used.

[0263] (19) Cell Culture and Liposome Binding

[0264] A monolayer culture of murine macrophage-like cells (J774A.1)obtained from Riken Cell Bank (Tsukuba) was maintained in RPMI 1640medium supplemented with 10% fetal carf serum (FCS). For bindingexperiments, the cells (8×10⁵ cells/mL, RPMI1640) were dispensed in anamount of 1 mL/well into a 12-well culture plate (Sumitomo Bakelite Co.,Ltd.) and were incubated for 24 hours at 37° C., then the culture brothwas replaced with Celgrosser-P medium (Sumitomo Pharmaceutical Co.).After one hour of preincubation at 37° C., 50 μL of liposomes (50 nmollipid/well) with or without β₂-GPI (200 μg/mL) and WB-CAL-1 (100 μg/mL)were added to each culture, and the cells were incubated at 4° C. and/or37° C. The wells were next washed with chilled PBS, and the cells werelysed by adding 1 mL of 0.1N NaOH. An aliquot was taken fordetermination of cellular proteins and of radioactivity associated withthe cells. Protein concentration was determined in a manner as describedabove.

[0265] (20) Anti-β₂-GPI ELISA (ELISA of IgG Autoantibody Against β₂-GPI)

[0266] Anti-β₂-GPI ELISA (ELISA of IgG autoantibody against β₂-GPI) wasperformed through a method as described in [43]. Briefly, β₂-GPI (10μg/mL, 50 μL/well) was adsorbed on an oxygenated polystyrene plate(carboxylated, Sumilon C-type, Sumitomo Bakelite Co., Ltd.) byincubating overnight at 4° C. The plates were blocked with 3% gelatin,and 100 μL/well of anti-β₂-GPI monoclonal antibody or of 100-folddiluted plasma samples was added to the plate, followed by incubationfor one hour. A antibody binding to β₂-GPI was probed using HRP-labeledanti-human IgG or IgM or anti-mouse IgG. The color was developed withH₂O₂ and o-phenylenediamine, and OD was measured at 490 nm. Betweenthese steps, extensive washing were done using PBS containing 0.05%Tween 20.

[0267] (21) ELISA for Antibodies Against a Protein-oxLig-1 Complex

[0268] Synthesized oxLig-1 (50 μg/mL, 50 μL/well) was adsorbed byevaporation on a polystyrene plate (Immulon 1B; Dynex TechnologiesInc.), and the plate was then blocked with 1% BSA. Serum samples(diluted 1:100 with PBS containing 0.3% BSA) were incubated in the wellswith β₂-GPI (15 μg/mL) or other proteins for one hour at roomtemperature. Antibody binding was detected with HRP-labeled anti-humanIgG. Further steps were performed through a method as described in“anti-β₂-GPI ELISA.”

[0269] (22) β₂-GPI-Dependent aCL

[0270] CL (50 μg/mL, 50 μL/well) was adsorbed on a polystyrene plate(Immulon 1B), and further steps were performed through a method asdescribed in “ELISA for antibodies against a protein-oxLig-1 complex.”

[0271] (23) Statistical Analysis

[0272] Statistical analysis was performed by use of StatView software(product of Abacus Concepts). Comparative studies between theautoantibody and the clinical episode were carried out by means of theFisher's exact test.

[0273] The relationship between the antibody value and the clinicalepisode was evaluated by means of the Mann-Whitney U-test.

<2> Results

[0274] (1) Molecular Interaction

[0275] oxLDL but not native LDL showed highly specific binding to β₂-GPI(FIG. 1A). A dose-dependent binding of oxLDL to solid phase WB-CAL-1 wasobserved only in the presence of β₂-GPI (10 μg/mL), and no specificbinding of native LDL was observed (FIG. 1B). In a control experiment,CL showed large extent of binding, while DOPE did not show any specificbinding (FIG. 1C). CL also showed dose-dependent binding to WB-CAL-1 inthe presence of β₂-GPI, while DOPE did not (FIG. 1D).

[0276] (2) Binding of β₂-GPI and Anti-β₂-GPI Antibody to Solid PhaseLDLs or Lipids Derived therefrom

[0277] β₂-GPI specifically bound with high avidity to immobilized oxLDL,but minimally to native LDL (FIG. 2A). Lipids were extracted from LDLs,immobilized on a plate, and subjected to binding assays for β₂-GPI bydetecting with Cof-22 monoclonal antibody and anti-β₂-GPI antibodies(i.e., WB-CAL-1 and EY2C9). The assays showed specific binding of β₂-GPI(FIG. 2B) and of β₂-GPI-mediated antibody to the lipids derived fromoxLDL (FIGS. 2C and 2D), but did not to those derived from native LDL(FIGS. 2B-2D).

[0278] (3) Purification and Characterization of a β₂-GPI-Specific Ligand

[0279] The lipids extracted from each LDL were spotted on a TLC plateand developed in solvent A (FIGS. 3A and 3B). In the plates treated withI₂ vapor and molybdenum blue, decreased PC (phosphatidylcholine) andincreased polar forms co-migrating with lysoPC were observed in lipidsderived from oxLDL as compared with derived from of native LDL. Bystaining with orcin/sulfuric acid, pseudo-positive bands were observedat similar Rf positions of Chol, PC, and CL, and others. InLieberman-Burchard reaction, a Chol band was observed for both extractsat almost top, and few bands near the Rf position of CL were observedfor lipid derived from oxLDL. To identify β₂-GPI-specific ligands, thedeveloped plates were subjected to ligand blot in which the plates weretreated with β₂-GPI and anti-β₂-GPI antibodies (FIG. 3B). With all threetested anti-β₂-GPI antibodies (Cof-22, WB-CAL-1, and EY2C9), twopredominant bands and several bands of diffused lipids were stained andobserved at similar Rf positions of CL and glycolipids such asgalactosylceramide (Gal-Cer) and glucosylceramide (Glc-Cer). The bandsdetected in the ligand blot were not stained with a spray of molybdenumblue (FIGS. 3A and 3B).

[0280] β₂-GPI-ligand-enriched lipids were scraped from the first TLCplate (hereinafter referred to as “scraped lipids”) (FIG. 3B) and weresubjected to another TLC (developing in solvent B), and the two majorbands were scraped off and subjected to the ligand blot (FIGS. 3C and3D). Two major bands (indicated by arrows in FIG. 3) reacting withβ₂-GPI were named Band-1 (the upper band) and Band-2 (the lower band).

[0281] Band-1 was rarely stained in the sequential treatment with β₂-GPIand EY2C9 (or WB-CAL-1) (2-step), but was strongly stained in the caseof the simultaneous treatment (1-step). In contrast, Band-2 was stainedwell in either treatment with β₂-GPI and EY2C9 (Densitometric analysisin individual experiments indicated significant difference).

[0282] The alkaline hydrolyzed (20% NaOH, 100° C., 30 minutes) productof Band-1 did not bind to β₂-GPI in the ligand blot (data not shown).Band-1 was subjected to reversed phase HPLC by use of solvent C. A majorpeak appeared at 22 min that positively stained in the ligand blot withβ₂-GPI and EY2C9 (FIG. 4), and was attributable to very weakLieberman-Burchard reaction. This peak was designated as oxLig-1. From100 mg protein equivalent of oxLDL, approximately 2.5 mg of oxLig-1 wasrecovered.

[0283] (4) Purification of oxLig-2

[0284] The oxLig-2, which is a specific ligand to β₂-GPI, was purifiedfrom a ligand-containing fraction through reverse phase HPLC by use of aSephasil Peptide C18 column (4.6 mm×250 mm, product ofAmersham-Pharmacia Biotech). The thus-separated Band-2 was eluted at aflow rate of 0.5 mL/min by 0.2% acetic acid-containing water (solvent E)and acetonitrile/isopropanol (30/70, v/v) (solvent D) with lineargradation (50-100%) in concentration for 15 minutes and then by 100%solvent D for 15 minutes. The absorbance (A) at 210 nm or at 234 nm wasmonitored. The eluate was fractionated (1 mL/tube) every two minutes.Each fraction was applied to a TLC plate and subjected to ligand blotanalysis using β₂-GPI and EY2C9.

[0285] (5) Purification and Properties of oxLig-2

[0286] Through HPLC of Band-2, a novel ligand (oxLig-2) was obtained(FIGS. 16A and 16B).

[0287] The peaks corresponding to specific binding to β₂-GPI and EY2C9were observed at about 26.7 min (equivalent to elution volume of 13.4mL). In order to confirm the purification degree of oxLig-2 (fraction14), the fraction was subjected to HPLC again under the same conditions(FIGS. 16C and 16D), and to an LC/MS analysis.

[0288] In a positive ionization mass spectrum of oxLig-2, three signals(m/z: 383, 441, and 627) were detected (FIG. 17C). Two small peaks;i.e., m/z of 383 (attributable to 7-ketocholesterol) and of 441(attributable to 7-ketocholesterol (+acetone)) are similar to thoseobserved in the case of ox-Lig-1 and 13-COOH-7-KC (FIGS. 17A, 17C, and17E).

[0289] The signals of 571, 627, and 627 (m/z) shown in the positiveionization mass spectra of oxLig-1, oxLig-2, and 13-COOH-7KC,respectively, were identified as respective parent ions [M+H]+ (FIGS.17A, 17C, and 17E). The signals of 569, 625, and 625 (m/z) shown in thenegative ionization mass spectra of oxLig-1, oxLig-2, and 13-COOH-7KC,respectively, were identified as respective parent ions [M+H]− (FIGS.17B, 17D, and 17F). In the spectrum of oxLig-2, a signal of 627 (m/z)was identified as a parent ion of dihydro-oxLig-2 (FIG. 17D). In thenegative mode, the signals of 187, 243, and 243 (m/z) shown in thespectra of oxLig-1, oxLig-2, and 13-COOH-7KC, respectively, wereidentified as respective fragment ions [D−H]− (FIGS. 17B, 17D, and 17F,and FIG. 18).

[0290] In all TLC ligand blot analyses using solvent A or B, the Rfposition with respect to oxLig-2 is lower than that of the relevantmonocarbonyl derivatives (oxLig-1 and 13-COOH-7KC). The featurecoincides with the presumed difference in polarity (FIGS. 3, 8, and 15).

[0291] In the TLC ligand blot analysis using solvent B, the Rf positionsin the bands with respect to oxLig-2 and 13-COOH-7KC methylated bydiazomethane (Me-oxLig-2 and Me-13-COOH-7KC, respectively) were higherthan that of the relevant unmethylated ligands (FIGS. 8 and 15). Thepeak corresponding to oxLig-2 (26.7 min) in reverse phase HPLC wasobserved earlier than oxLig-1 (27.3 min) and 13-COOH-7KC (28.9 min). Thepeaks corresponding to methylated oxLig-2 and methylated 13-COOH-7KCwere observed (27.1 min and 30.0 min, respectively) later than those ofthe unmethylated species. Surprisingly, in TLC ligand blot analyses, theinteraction of β₂-GPI and the anti-β₂-GPI antibodies (Cof-22 and EY2C9)with each ligand was completely suppressed through ligand methylation.

[0292] In ELISA of the anti-β₂-GPI antibodies by use of a ligand-coatedplate, significant binding of the anti-β₂-GPI autoantibodies (WB-CAL-1and EY2C9) to solid-phase oxLig-1, oxLig-2, and 13-COOH-7KC wereobserved, but no such binding was observed with respect to solid-phasemethylated oxLig-2, methylated 13-COOH-7KC, or control lipid(cholesteryl linoleate). Similar results were obtained in the case wherea mouse monoclonal anti-β₂-GPI antibody obtained from mice immunizedwith human β₂-GPI was used (Table 1). These three antibodies did notbind to the immobilized cholesterol or to 7-ketocholesterol. From theseresults and previously reported results [52], it is concluded that thestructure of oxLig-2 is highly likely to be an oxide of cholesteryllinoleate, 4,12-dioxo-12-(7-ketocholest-5-en-3β-yloxy)dodecanoic acid(FIG. 18). TABLE 1 Solid-phase lipid β2-GPI binding (Cof-22 binding)WB-CAL-1 binding EY2C9 binding (Ligand) No treatment Methylation Notreatment Methylation No treatment Methylation Cholesteryl 0.061 +/−0.005 N.T. 0.056 +/− 0.002 N.T. 0.074 +/− 0.012 N.T. linoleate oxLig-11.307 +/− 0.105 N.T. 0.945 +/− 0.068 N.T. 1.458 +/− 0.062 N.T. oxLig-21.002 +/− 0.084 0.204 +/− 0.029 0.518 +/− 0.023 0.106 +/− 0.018 1.018+/− 0.121 0.076 +/− 0.018 13-COOH-7KC 1.303 +/− 0.049 0.094 +/− 0.0020.336 +/− 0.027 0.077 +/− 0.002 1.062 +/− 0.040 0.052 +/− 0.000

[0293]FIG. 15 shows the results of ligand blot with respect to oxLig-2,Me-oxLig-2, 13-COOH-7KC, and Me-13-COOH-7KC. As is clear from FIG. 15,reactivity of β₂-GPI and that of the β₂-GPI antibody is lost throughmethylation of oxLig-2 and 13-COOH-7KC.

[0294] oxLig-1 was further analyzed through LC/MS. The intensities ofboth positive and negative ions attributed to oxLig-1 were detected forthe main peak (at 8.3 min) at 234 nm (FIG. 5A). A positive ionizationmass spectrum gave a signal at m/z 571, which was considered to beattributed to (M+H)⁺, and at m/z 383 (FIG. 5D), which was identical tothat attributed to ionized 7-ketocholesterol (FIG. 5B).

[0295] In the negative ion mode, a signal of fragment ion was detectedat m/z 187 (FIG. 5C). oxLig-1 was treated with diazomethane in diethylether to give methylated oxLig-1, which was analyzed through LC/MS.Methylated oxLig-1 was eluted later than oxLig-1 (FIG. 5A, lower), andsignals of the largest fragment ion (9.0 min) was detected at m/z 585and at m/z 201, in the positive and negative ion modes, respectively(FIGS. 5E and 5F). These data are consistent with the structure of9-oxo-9-(7-ketocholest-5-en-3β-yloxy)nonanoic acid.

[0296] (6) Synthesis and Analysis of oxLig-1

[0297] In order to confirm the structure of oxLig-1, oxLig-1 wassynthesized from 7-ketocholesterol and azelaic acid. As shown in FIG. 6,the materials were processed with WSC and DMAP in acetone at roomtemperature for two days, whereby both materials were conjugated eachother. The product was isolated through column chromatography on silicagel. The structure of the synthesized oxLig-1 was verified to purifiedoxLig-1 through ¹H- and ¹³C-NMR spectroscopy and FD mass spectrometry.In the ¹H-NMR spectrum of synthesized oxLig-1 (synthesized oxLig-1)(FIG. 7A), the signals of H-3 and H-6 are shown at δ 4.69-4.78 and 5.71ppm as multiplet and singlet, respectively. Furthermore, three peaksassignable to carbonyl carbons are shown at δ 202.5, 179.7, and 173.4ppm together with two signals of olefin carbons at δ 164.5 and 127.1 ppm(FIG. 7B).

[0298] The synthesized oxLig-1 was then esterified with diazomethane indiethyl ether to give methylated oxLig-1. In FIGS. 7C and 7D, by ¹H- and¹³C-NMR spectra of synthesized oxLig-1 are shown. The new singlet wasobserved in its ¹H-NMR spectrum at δ 3.67 ppm, strongly suggesting thatoxLig-1 has a carboxyl group (FIG. 7C). Then, synthesized oxLig-1 wassubjected to TLC and ligand blot analysis with β₂-GPI and anti-β₂-GPIantibodies. Synthesized oxLig-1 showed the same Rf position and bindingcharacteristics to Cof-22, WB-CAL-1, and EY2C9 antibodies, as previouslydescribed for oxLig-1 derived from oxLDL (FIGS. 3 and 8).

[0299] The synthesized oxLig-1 and its methylated compound were furtheranalyzed through LC/MS. The LC chromatograms and mass spectra of bothcompounds (FIG. 9) were identical to the corresponding compounds derivedfrom oxLDL (FIG. 5). In FIG. 9D, the small peak at 8.3 min is attributedto synthesized oxLig-1 that remained underivatized after the methylationreaction. The underivatized material was identified as synthesizedoxLig-1 through mass spectrometry.

[0300] (7) Liposome Binding to Macrophages

[0301] Binding to the J774A.1 cells of exogenous PS-Chol-liposomesincreased depending on the amount of DPPS (FIG. 10). In contrast,binding to the cells of oxLig-1-Chol-liposomes was relatively low.Similar binding profiles were obtained with Chol-free liposomes of PS oroxLig-1. When mouse peritoneal macrophages were used in place of J774A.1cells, comparable liposome binding was observed.

[0302] (8) Antibody-Dependent Liposome Binding to Macrophages

[0303] Binding (4° C., 2 hours) of PS-liposomes to oxLig-1-liposomesincreased dramatically upon simultaneous addition of β₂-GPI andWB-CAL-1, and was also dependent on the concentration of WB-CAL-1 (FIGS.11A and 11B). In the same assay, subclass-matched control antibodies hadno effect on such binding. The uptake (37° C., 5 hours) ofoxLig-1-liposomes by J774A.1 cells increased significantly by incubating(4.36 pmol [³H]DPPC/mg protein) with β₂-GPI and WB-CAL-1, as compared toincubation without β₂-GPI and WB-CAL-1 (0.72 pmol [³H]DPPC/mg protein)(data not shown). As shown in FIG. 11C, binding of synthesizedoxLig-1-liposomes to the macrophages also increased depending on theligand concentration in liposomes. In the case of synthesized oxLig-1,the binding reached almost plateau at the concentration of 10 mL %.

[0304] (9) Binding of Liposomes Containing oxLig-2, 13-COOH-7KC, or theLike to Macrophage

[0305] Direct binding of liposomes containing oxLig-1, oxLig-2, or13-COOH-7KC to macrophages, J774A.1 cells, was compared with that ofliposomes containing DPPS. DPPS-containing liposomes exhibited bindingto the macrophages with ligand concentration dependency. In contrast,binding of liposomes containing oxLig-1, oxLig-2, or 13-COOH-7KC tomacrophages was relatively low or negligible small (FIG. 19). As isclear from FIG. 19, scavenger receptors are not related to binding ofthese liposomes to macrophages except the case of DPPS-containingliposomes. In other words, uptake of liposomes containing oxLig-1,oxLig-2, or 13-COOH-7KC to J774A.1 cells was significantly enhanced inthe presence of both β₂-GPI and an anti-β₂-GPI antibody (WB-CAL-1), ascompared with cholesteryl linoleate-liposomes (control) (FIGS. 20A, 20B,20C, and 20D). In contrast, substantially no binding of liposomes wasobserved after methylation of oxLig-2 or 13-COOH-7KC (FIGS. 20C and20D).

[0306] (10) Detection of Autoantibodies in APS Patients with Episodes ofArterial Thrombosis through ELISA Using a β₂-GPI-Synthesized oxLig-1Complex.

[0307] As shown in FIG. 12, autoantibodies against a complex of β₂-GPIand synthesized oxLig-1 were detected in APS patients at high frequency.There was a good correlation between values of autoantibodies againstthe complex antigen and those of β₂-GPI-dependent aCL or anti-β₂-GPIantibodies. So far as the tests were preformed, such autoantibodiesderived from APS cross-reacted with β₂-GPI complexed with CL or oxLig-1,but not that complexed with oxidized PAPC (oxPAPC) (Table 2, Exp-1). Theantibody binding did not correlate with the amount of TBARS in lipid.Further, the antibody binding was provided only by the interactionbetween oxLig-1 and intact β₂-GPI (Exp-2). In contrast, no antibodybinding was provided by nicked β₂-GPI or haptoglobin having “sushidomains” (which did not have PL binding property). TABLE 2 Experiment 1Solid-phase lipid Binding of β₂-GPI-dependent aCL (Absorbance, mean ±SD, n = 3) (TBARS)^(a) Cof-22 EY2C9 WB-CAL-1 APS-1^(b) APS-2^(b) CL(4.46) 1.132 ± 0.025 1.269 ± 0.014 1.361 ± 0.008 2.099 ± 0.216 1.282 ±0.041 Synthesized 1.066 ± 0.114 0.915 ± 0.072 1.035 ± 0.062 1.130 ±0.177 1.222 ± 0.057 oxLig-1 (0.66) PAPC (107.8) 0.019 ± 0.003 0.011 ±0.003 0.012 ± 0.001 0.000 ± 0.001 0.010 ± 0.002 oxPAPC^(c) (218.3) 0.073± 0.007 0.009 ± 0.002 0.015 ± 0.005 0.007 ± 0.002 0.015 ± 0.001Experiment 2 Binding of protein-dependent oxLig-1 antibody (Absorbance,mean ± SD, n = 3) Added protein Cof-22 EY2C9 WB-CAL-1 APS-1^(b)APS-2^(b) w/o 0.050 ± 0.002 0.005 ± 0.001 0.005 ± 0.002 0.012 ± 0.0030.014 ± 0.001 β₂-GPI 1.007 ± 0.031 1.147 ± 0.028 0.812 ± 0.023 1.043 ±0.054 0.755 ± 0.024 Nicked β₂-GPI 0.164 ± 0.007 0.005 ± 0.001 0.003 ±0.001 0.008 ± 0.003 0.011 ± 0.001 Haptoglobin 0.049 ± 0.001 0.006 ±0.001 0.006 ± 0.001 0.018 ± 0.011 0.015 ± 0.002 Ovalbumin 0.042 ± 0.0010.004 ± 0.002 0.006 ± 0.002 0.009 ± 0.004 0.016 ± 0.001

[0308] Table 2 shows the results of binding of anti-β₂-GPIantiphospholipid antibodies (Abs) to lipid-proptein complexes observedthrough enzyme-linked immunosorbent assay (ELISA).

[0309] Data shown in Experiment 1 represent binding of antibodies in thepresence of β₂-GPI (15 μg/mL), and data shown in Experiment 2 representthe results of ELISA performed by use of a synthesizedoxLig-1-solidified plate in the presence of each of the proteins (15μg/mL) shown in Table 2. The amount of TBARS (thiobarbituric acidreactive substance) shown in the Table is based on nmol (malondialdehydeequivalent)/mg (lipid). APS-1 and APS-2 were obtained from serum samplesof APS patients having an episode of arterial thrombosis with removal ofβ₂-GPI. oxPAPC was obtained by exposing PAPC in air at room temperaturefor 24 hours. Nicked β₂-GPI was prepared through plasmin treatment.[43].

[0310] (11) The following results were obtained through further analysisof the relationship between the anti-β₂-GPI-oxLig-1 antibody value andclinical observations. When the antibody value (OD) of a plasma sampleexceeded the averaged (30 healthy volunteers) antibody value by morethan 3×SD (standard deviation), the sample was regarded to be positiveto a specific antibody (as a cut-off value).

[0311] The IgG anti-β₂-GPI-oxLig-1 antibody was observed in 73% theinitial-stage APS patients (35/48); in 59% the APS patients with SLE(second-stage APS) (23/39); and in 11% the solo-SLE patients (5/46)(Table 3). TABLE 3 Patient No. % SLE only 46 APS 87 Primary 48 55Secondary 39 45 Clinical profile Thrombosis 75 56 Arterial thrombosisonly 27 20 Venous thrombosis only 29 22 Arterial and venous thrombosis19 14 Pregnancy morbidity 32/120 27 Thrombocytopenia 24/128 19Autoantibody β₂-GPI-dependent aCL 77/133 58 (anti-β₂-GPI-CL antibody)Anti-β₂-GPI antibody 48/133 36 Anti-β₂-GPI-oxLig-1 antibody 63/133 47Lupus anticoagulants 62/113 55

[0312] In all patients (133 patients) investigated in the presentexample, the anti-β₂-GPI-oxLig-1 antibody value was found to be stronglycorrelated with the antibody value of the β₂-GPI-dependent aCL(anti-β₂-GPI-CL antibody) and that of the anti-β₂-GPI antibody(correlation factor r²: 0.72 and 0.81, respectively) (FIG. 14).

[0313] In these patients (133 APS and/or SLE patients), significantrelationship was observed between the anti-β₂-GPI-oxLig-1 antibody valueand episodes of thrombosis (arterial and/or venous thrombosis, arterialthrombosis, and venous thrombosis) or pregnancy morbidity, but nosignificant relationship was observed between the antibody value andthrombocytopenia (p=4.2×10⁻⁸ (Fisher's exact test); Odds ratio 8.15,p=1.7×10⁻⁷; 8.00, p=0.018; 2.29, P=0.0077; 3.03, and P=0.31; 1.38,respectively) (Table 4). Assay sensitivity, specificity, and expectedvalues with respect to arterial thrombosis diagnosis were remarkablyexcellent as compared with diagnosis of venous thrombosis, pregnancymorbidity, and thrombocytopenia (Table 4). TABLE 4 Thrombosis Arterialthrombosis Venous thrombosis Pregnancy morbidity Thrombocytopenia Auto-Odds Odds Odds Odds Odds antibody + − p* ratio + − p* ratio + − p*ratio + − p* ratio + − p* ratio Positive 51 12 4.2 × 10⁻⁸ 8.15 36 27 1.7× 10⁻⁷ 8.00 29 34 0.018 2.29 21 34 0.0077 3.03 13 48 0.31 1.38 Negative24 46 10 60 19 51 11 54 11 56 (NS) Specificity 0.79 0.69 0.60 0.61 0.54Sensitivity 0.68 0.78 0.60 0.63 0.54 Expected 0.73 0.72 0.60 0.63 0.54Value

[0314] In addition, as compared with the antibody value of theβ₂-GPI-dependent aCL and that of the anti-β₂-GPI antibody, theanti-β₂-GPI-oxLig-1 antibody value was more strongly correlated withthrombosis (arterial and/or venous thrombosis) (p=1.5×10⁻⁶ (Fisher'sexact test); Odds ratio 6.02, p=5.2×10⁻⁵; 4.93, respectively).

[0315] The anti-β₂-GPI-oxLig-1 antibody values of the patients having anepisode of thrombosis (arterial and/or venous thrombosis, arterialthrombosis, or venous thrombosis) or a pregnancy-related disease wassignificantly higher than that of subjects having no such an episode(Mann-Whitney U-test, p<0.0001, p<0.0001, P=0.025, and P=0.011) (FIG.15). In contrast, no relationship was observed between the presence ofthe antibody or the antibody value and the episode of thrombocytopenia(Mann-Whitney U-test, p=0.45).

<3> Discussion

[0316] A strong evidence for specific binding interactions among β₂-GPI,oxLDL, and an anti-β₂-GPI autoantibody, was obtained by use of anoptical biosensor. A ligand specific to β₂-GPI was purified, and itsstructure and involvement in macrophage uptake of oxLDL wascharacterized by using a synthesized ligand. The structure of the ligandwas confirmed by reproducing its properties with chemically synthesized9-oxo-9-(7-ketocholest-5-en-3β-yloxy)nonanoic acid.

[0317] It appears that oxidation of LDL plays an important role inatherogenesis [44]. To examine mechanisms related to the development ofatherosclerosis, several experimental models of denatured LDL, such asMDA-LDL, acetylated LDL, and CuSO₄-mediated oxLDL (CuSO₄-oxLDL), havebeen used. Among these LDLs, Cu²⁺-ion can induce LDL oxidation,resulting in highly reproducible LDL damage [45]. This process leads toproduction of oxLDL that shares many structural and functionalproperties with LDL oxidized by cells or LDL extracted from arterialatherosclerotic plaques. Incubation of LDL with several different typesof cells or incubation of LDL with Cu²⁺-ion even in the absence of cellsresult in generating oxidatively modified LDL with similar properties[46]. There is general agreement about the use of CuSO₄-oxLDL as anautoantigen because oxLDL has been found in atheromatous lesions andoxLDL extracted from atherosclerotic lesions exhibits nearly all of thephysicochemical and immunological properties of CuSO₄-oxLDL [24].

[0318] Antibodies against oxLDL recognize substances in atheroscleroticlesions that are not present in normal arteries. It has been reportedthat aCL raised in SLE patients cross-reacted with MDA-LDL [27], whileother research groups found that another population of anti-oxLDLantibodies reacted to oxidized PC (such as POVPC)-protein adducts in LDLmolecules [33, 47].

[0319] However, recent studies elucidated that TBARS generation was notconsistent with β₂-GPI binding to CuSO₄-oxLDL [48].

[0320] As MDA is a hydrophilic short-chain aldehyde, it readily diffusesaway from LDL particles [49]. It was also observed that, after dialysis,TBARS in the oxLDL preparations decreased to undetectably low levels. Inthis study, CuSO₄-oxLDL showed highly specific binding to immobilizedβ₂-GPI (FIG. 1A). The weak binding of native LDL to β₂-GPI might reflectthe binding of β₂-GPI to lipoprotein [a], which is composed of LDL andapolipoprotein [a] (apo[a]), as described in [50]. However, suchinteraction between native LDL and β₂-GPI may not expose suitableepitopes to anti-β₂-GPI autoantibody (FIG. 1B). Further, highly specificbinding of anti-β₂-GPI autoantibody, two monoclonal antibodies, andantibodies in two typical anti-β₂-GPI positive serum samples (derivedfrom APS patients with episodes of arterial thrombosis) was onlyobserved in the presence of the complex of β₂-GPI and the lipid ligandderived from CuSO₄-oxLDL (FIGS. 2, 3, and 12, Table 2). Oxidativemodification of LDL actually includes a series of complex changes suchas lipid peroxidation, modification of side chains of amino acids byactive aldehydes, increased surface charge, and polymerization.

[0321] Among diverse modified molecules in oxLDL, β₂-GPI obviously boundto a component in the lipid moiety.

[0322] Linoleic acid is a predominant polyunsaturated fatty acid in LDLand is present mainly as Chol-ester [51].

[0323] In mildly oxidized LDL, cholesteryl hydroperoxyoctadecadienoate(Chol-HPODE) and cholesteryl hydroxyoctadecadienoate (Chol-HODE) weredetected as main constituents of oxidation products [52]. Chol-HPODE hasbeen reported to inactivate platelet-derived growth factor [53].7-Hydroxycholesterol (both free and esterified) is the major oxysterolformed in an earlier stage of LDL oxidation, with 7-ketocholesteroldominating at later stages [54]. Recent studies indicated that elevatedplasma levels of 7β-hydroxycholesterol may be associated with anincreased risk of atherosclerosis [55]. At later stages in LDLoxidation, cholesteryl esters or 7-ketocholesteryl esters of9-oxononanoate derived from cholesteryl linoleate [56] were detected asthe most abundant fraction of oxidized cholesteryl linoleate [57, 58].Cholesteryl ester core-aldehydes react with the free ε-amino group oflysines, form complexes with proteins, and were evident in humanatheroscleorotic lesions [58, 59]. In the present study, oxLig-1 wasidentified to be 9-oxo-9-(7-ketochlest-5-en-3β-yloxy)nonanoic acid, oneof the oxidation products of cholesteryl linoleate, and a major ligandfor β₂-GPI. However, it still remains to be elucidated whether9-oxo-9-(7-ketochlest-5-en-3β-yloxy)nonanoic acid is actually present inbiological samples as an oxLDL ligand.

[0324] Chemically modified LDL can be rapidly taken up by macrophagesvia receptor-mediated endocytosis, resulting in foam cell formation[44]. As models of oxLDL, oxLig-1- and PS-liposomes were used to studytheir binding to J774A.1 cells (FIG. 7). PS-liposomes bound tomacrophages via scavenger receptor(s), while oxLig-1 did not seem to bea major ligand for scavenger receptors. However, the binding ofoxLig-1-liposomes to J774A.1 cells at 4° C. increased up to 14 timeswhen oxLig-1-liposomes were added simultaneously with β₂-GPI andWB-CAL-1. The uptake of oxLig-1-liposomes with J774A.1 cells at 37° C.also significantly increased by incubation with β₂-GPI and WB-CAL-1.This binding and uptake might be mediated by the Fcy receptor. Theuptake by macrophages of immune complexes containing oxLDL through theFcγ type I receptor transformed macrophages into foam cells [60, 61],and could accelerate the atherogenic process [62-64].

[0325] Autoantibodies against a solid phase oxLig-1 complexed withβ₂-GPI were detected in serum samples collected from APS patients havingepisodes of arterial thrombosis (FIG. 12). Further, there was a goodcorrelation between anti-β₂-GPI-oxLig-1 antibody value, anti-β₂-GPIantibody value, and β₂-GPI-dependent aCL titer. In contrast, it has beenreported that some aPL recognize adducts of oxidized PLs and β₂-GPI[31]. It has not been determined whether oxLig-1can form covalentadducts with β₂-GPI, and the possibility can not yet be excluded.However, it is clear that interaction between oxLig-1 and β₂-GPI isessential to express antigenicity for the autoantibodies shown inTable-2, Exp-2. It was also suggested that domain V, which contains the-PL-binding region [15, 16], is distinguished from domains in whichepitopes, recognized by aPL in APS patients, locate [38, 65].

[0326] In addition to promoting lipid deposition in macrophages, oxLDLis considered to have another characteristic that it may accelerateatherogenesis. oxLDL is chemotactic for monocytes and for T cells [66,67] and is cytotoxic for cultured endothelial cells [68]. It has beenalso reported that peroxisome proliferator-activated receptor γ (PPARγ),a transcriptional regulator of genes linked to lipid metabolisms, isactivated by components of oxLDL, such as 9-HODE, and 13-HODE and CD36,a scavenger receptor, is up-regulated by a combination of PPARγ andretinoid X receptor ligands [69]. PPARγ enhances the uptake of oxLDL,thereby promoting foam cell formation. It remains to be determined ifoxLig-1 has any specific influence on nuclear receptors or otherbiological functions such as intracellular signal transductions.

[0327] In summary, a ligand for β₂-GPI present in oxLDL was isolated andcharacteirzed. The ligand (oxLig-1), i.e.,9-oxo-9-(7-ketochlest-5-en-3β-yloxy)nonanoic acid, mediates liposomeuptake by macrophages in the presence of β₂-GPI and an anti-β₂-GPIautoantibody. These findings on the ligand provide a specific structuraland mechanistic link between β₂-GPI and anti-β₂-GPI autoantibodies andatherogenesis in APS.

[0328] The ω-carboxyl group introduced through oxidation by Cu²⁺ wasindicated to play an important role in interaction between β₂-GPI andthe ligand. The ligand, oxLig-2, was identified as4,12-dioxo-12-(7-ketocholest-5-en-3β-yloxy)dodecanoic acid (FIG. 18).

[0329] Although results of methylation of oxLig-2 indicated that thecarboxylic group was present in the acyl chain, the accurate position ofthe ketonic moiety cannot be determined through mass spectroscopy.However, since cholesteryl linoleate is one of the important cholesterylesters of LDL [52], the positions of the ketonic moieties are highlylikely 9-position and/or 13-position.

[0330] β₂-GPI did not bind to cholesterol, 7-ketocholesterol, orcholesteryl linoleate, but did form significant bonding to oxLig-1,oxLig-2, or 13-COOH-7KC. Thus, the formed oxysterol esters having acarboxylated long acyl chain constitute a novel class of amphipathicligands suitable for β₂-GPI. Furthermore, the fact that methylation ofthese ligands inhibits interaction between the ligands and β₂-GPIindicated requirement of a free carboxylic group for structurerecognition.

[0331] The results of experiments indicated that oxidized cholesterylesters, particularly such esters having 7-ketocholesterol and a carboxylgroup in the acryl group functioned as ligands to β₂-GPI and ananti-β₂-GPI autoantibody. One predominant biologically oxide compoundoriginating from plasma LDL may be a ω-carboxylated oxysterol such asoxLig-1 or oxLig-2. 13-COOH-7KC, which is an artificially synthesizedcompound, also formed significant bonding to β₂-GPI, similar to thecases of oxLig-1 and oxLig-2.

<4> Fabrication of the Kits of the Present Invention

[0332] The kit 1 of the present invention containing the followingelements was prepared:

[0333] 1. A 96-well immuno-plate on which oxLig-1 has been immobilized(1 sheet);

[0334] 2. β₂-GPI standard solution (1 set);

[0335] 3. Anti-β₂-GPI antibody (WB-CAL-1) (1 vial);

[0336] 4. HRP-labeled anti-mouse IgG antibody (1 vial);

[0337] 5. o-Phenylenediamine solution (1 vial);

[0338] 6. Aqueous hydrogen peroxide (1 vial); and

[0339] 7. Reaction-terminating liquid (1N HCl) (1 vial).

[0340] The kit 2 of the present invention containing the followingelements was prepared:

[0341] 1. A 96-well immuno-plate on which oxLig-1 has been immobilized(1 sheet);

[0342] 2. β₂-GPI (1 vial);

[0343] 3. HRP-labeled anti-human IgG antibody (1 vial);

[0344] 4. Tetramethylbenzidine solution (1 vial);

[0345] 5. Aqueous hydrogen peroxide (1 vial); and

[0346] 6. Reaction-terminating liquid (1N HCl) (1 vial).

<5> References

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[0387] 69. Cell. 93: 229-240

Industrial Applicability

[0388] The derivative of the present invention specifically binds toβ₂-GPI, and thus can be used for, for example, detecting or purifyingβ₂-GPI. The derivative can also be applicable to the solid phase of thepresent invention, the assay method of the present invention, thedetection method of the present invention, and the assay kit of thepresent invention. Thus, the derivative of the present invention isremarkably useful. Furthermore, oxLDL can be assayed making use of thederivative of the present invention through a competitive method and thederivative can be employed as a standard substance for oxLDL assay.

[0389] The solid phase of the present invention can be used, forexample, in simple, quick detection of β₂-GPI or purification of β₂-GPIas well as is applicable to the assay method of the present invention,the detection method of the present invention, and the assay kit of thepresent invention. Thus, the solid phase of the invention is remarkablyuseful.

[0390] Through the assay method 1 or 2 of the present invention, β₂-GPI,an autoantibody against a complex of β₂-GPI and the derivative of thepresent invention, or a similar substance can be assayed quickly in asimple manner. Thus, the assay methods 1 and 2 of the present inventionare remarkably useful.

[0391] Through the detection method of the present invention, a diseasecan be detected quickly in a simple manner. Thus, the detection methodof the present invention is remarkably useful.

[0392] By use of the assay kit 1 or 2 of the present invention, theassay method 1 or 2 can be carried out more quickly and in a simplermanner. Thus, the assay kits of the present invention are remarkablyuseful.

1. A cholesterol derivative represented by the following formula (1):

wherein R represents a C3-C23 saturated or unsaturated aliphatichydrocarbon residue having an optional substituent, and, to thecholesterol backbone, —OH, —CHO, —COOH, —OOH, or an epoxy group may beadded.
 2. The cholesterol derivative as described in claim 1, whereinthe hydrocarbon residue has one or more substituents selected from thegroup consisting of —COOH, —OH, —CHO, an oxo group, and an epoxy group.3. The cholesterol derivative as described in claim 1, wherein R is aC3-C23 saturated or unsaturated fatty acid residue which may have one ormore oxo groups.
 4. The cholesterol derivative as described in claim 1,wherein R is a group represented by HOOC—R′— (wherein R′ represents aC2-C22 saturated or unsaturated aliphatic hydrocarbon residue which mayhave one or more oxo groups).
 5. The cholesterol derivative as describedin claim 4, wherein R′ is a linear-chain aliphatic hydrocarbon residuehaving one oxo group.
 6. A cholesterol derivative represented by thefollowing formula (2).


7. A cholesterol derivative represented by the following formula (3).


8. A cholesterol derivative represented by the following formula (4).


9. A cholesterol derivative represented by the following formula (5).


10. A cholesterol derivative represented by the following formula (6).


11. A cholesterol derivative represented by the following formula (7).


12. A solid phase on which a cholesterol derivative as recited in claim1 has been immobilized.
 13. An assay method for β₂-GPI, characterized inthat the method includes at least the following steps: a step of forminga complex of β₂-GPI and the cholesterol derivative immobilized on asolid phase as recited in claim 12 by bringing a specimen into contactwith the solid phase (Step 1); and a step of detecting β₂-GPI containedin the complex which has been formed in Step 1 (Step 2).
 14. An assaymethod for an antibody recognizing a “complex of β₂-GPI and acholesterol derivative as recited in claim 1,” characterized in that themethod includes at least the following steps: a step of forming acomplex of a “complex of β₂-GPI and a cholesterol derivative immobilizedon a solid phase” and an antibody recognizing the “complex of β₂-GPI andthe cholesterol derivative immobilized on the solid phase” by bringingβ₂-GPI and a specimen into contact with the solid phase (Step 1); and astep of detecting the antibody contained in the complex which has beenformed in Step 1 (Step 2).
 15. A method for detecting a disease,characterized in that the method includes assaying an antibodyrecognizing the “complex of β₂-GPI and the cholesterol derivative asrecited in claim 1” present in blood and correlating the amount of theantibody present in blood to the disease.
 16. The method for detecting adisease as described in claim 15, wherein the disease is anantiphospholipid syndrome or thrombosis.
 17. The method for detecting adisease as described in claim 16, wherein the thrombosis is arterialthrombosis.
 18. An assay kit for β₂-GPI, characterized in that the kitcomprises at least the following (A) and (B): a solid phase as recitedin claim 12 (A) and a substance binding to β₂-GPI (B).
 19. An assay kitfor an antibody recognizing “a complex of β₂-GPI and a cholesterolderivative as recited in claim 1,” characterized in that the kitcomprises at least the following (A) and (B): a solid phase havingimmobilized thereon a cholesterol derivative as recited in claim 1 (A)and a substance binding to an antibody recognizing “the complex ofβ₂-GPI and the cholesterol derivative as recited in claim 1” (B). 20.The assay kit as described in claim 19, which is a kit for detecting adisease.
 21. The assay kit as described in claim 20, wherein the diseaseis an antiphospholipid syndrome or thrombosis.
 22. The assay kit asdescribed in claim 21, wherein the thrombosis is arterial thrombosis.